WP2: Biomarkers for acute myocardial infarction

The aim of this WP is to develop methods for the traceable quantification of cTn, an important biomarker for acute myocardial infarction, using species specific IDMS.
To achieve the goals of this WP, pure standard material as well as an isotopically enriched protein as spike material will be produced and characterised in Task 2.1 regarding the identity and modification of cTn using elemental and molecular MS and NMR. These materials will then be used as calibration and spike material, respectively, in developing an IDMS method. In Task 2.2 methods for the traceable quantification of cTn including the investigation of separation methods of the named protein will be developed. These methods are subsequently validated in Task 2.3 with special focus on possible interferences in human samples. Finally, the developed methods will be applied to clinical and EQA samples in Task 2.4.

As a first step in producing the isotopically enriched spike material, all subunits of cTn and the complex could be expressed successfully in E. coli and were isolated using affinity and size exclusion chromatography. Selenomethionine-labelled cTnC was successfully prepared and is available for shipment to the partners. A method for the separation and quantification of cTn via its sulphur content using LC coupled to ICP-MS has been developed. However, the sensitivity is not sufficient for clinical samples; so a new strategy using lanthanide labels is currently under investigation, either detecting the whole protein or specific peptides after enzymatic digestion. Additionally, the quantification method via specific peptides using LC-MS published by Schneck et al. (Anal Bioanal Chem 2018, 410:2805–2813) could be improved, so that complementary approaches will be available for mutual validation.
A conventional sandwich enzyme-linked immunosorbent assay (ELISA) has been established which will be used for the comparison of reference measurement procedure and routine methods. However, the sensitivity of the assay has still to be improved to be comparable to the high sensitivity assays in use at clinical laboratories nowadays. Furthermore, experience with different antibodies during the development of the ELISA could be used in the development of the biosensor for cTn.
 

A flow injection assay (FIA) – based biosensor setup (hardware and initial software) has been established and the assay has been tested on blood plasma. Different combination of antibodies has been tested to give the best sensitivity and selectivity. Still some interferences as well as sensitivity issues need to be resolved before the biosensor is fit-for-purpose to be used as point-of-care test.

Biosensor development 1

Biosensor development 2